Figure 2.
JQ1 and JQ5 impair the GC reaction in murine cGVHD/BO mice. (A-I) Transplants were performed as in Figure 1; groups are as defined in Figure 1. Results shown are pooled from 3 transplant replicates. (A-C) Flow cytometry analysis of mouse splenocytes taken 49 days posttransplant. TFH frequency is defined as % CXCR5+, PD1+ of FoxP3−, CD4+ live splenocytes. TFRs are the CXCR5+, PD1+ percentage of FoxP3+, CD4+ live splenocytes. The TFR/TFH ratio is shown. GCBs are GL7+, FAShi percentage of the CD19+ live splenocytes. (A) JQ5 treatment resulted in a significant decrease in TFH and GCB frequencies and increase in TFR frequency and the TFR/TFH ratio (n = 23/BM-only, n = 18/cGVHD, n = 15/JQ5). (B) Similar results are shown for each population with JQ1 treatment, consistent with reduced GC reaction (n = 14/BM-only, n = 11/cGVHD, n = 7/JQ1). (C) Representative gating for (left) TFH and TFR cells and (right) GCB cells. BM-only contours are in blue, cGVHD contours are in red. (D) Representative images of cryopreserved spleen sections stained to show GCs from mice 49 days posttransplant. Sections are stained with DAPI (blue), peanut agglutinin (PNA) rhodamine (red), and CD4 FITC (green). Images are at ×200 magnification. (E-F) (Left) Number of GCs observed in each spleen section normalized for the area of spleen in each section. A GC was counted if it was a roughly circular region of PNA+ cells near a region of CD4+ cells. Right: quantification of the average size of GCs observed in each section. The GC size was determined as the area of the PNA+ region. For both JQ5 (E; n = 8/BM-only, n = 6/cGVHD, n = 5/JQ5) and JQ1 (F; n = 5/group), there was a significant reduction in both GC count and average size. (G) Flow cytometric analysis of single-cell lung suspensions taken from transplanted mice treated with each drug (n = 5/group). Left: total plasma cells are CD138+ lymphocytes, (center) immature plasma cells are B220+, CD19+ plasma cells, and (right) mature plasma cells are B220−, CD19− plasma cells. Results show that both drugs significantly reduced the proportion of mature plasma cells in subject lungs. (H) Mean fluorescence intensity (MFI) quantification of flow cytometry analysis of H3K27me3 content of GC cell populations gated as in panel C (n per group as in panel A). (I) Representative histograms of GCB cells H3K27me3 content for BM-only, cGVHD, and JQ5-treated mouse-derived cells. For all panels, statistics shown are results of an unpaired t test with Bonferroni corrected P values, where appropriate. *P < .05, **P < .01, ***P < .001, ****P < .0001.

JQ1 and JQ5 impair the GC reaction in murine cGVHD/BO mice. (A-I) Transplants were performed as in Figure 1; groups are as defined in Figure 1. Results shown are pooled from 3 transplant replicates. (A-C) Flow cytometry analysis of mouse splenocytes taken 49 days posttransplant. TFH frequency is defined as % CXCR5+, PD1+ of FoxP3−, CD4+ live splenocytes. TFRs are the CXCR5+, PD1+ percentage of FoxP3+, CD4+ live splenocytes. The TFR/TFH ratio is shown. GCBs are GL7+, FAShi percentage of the CD19+ live splenocytes. (A) JQ5 treatment resulted in a significant decrease in TFH and GCB frequencies and increase in TFR frequency and the TFR/TFH ratio (n = 23/BM-only, n = 18/cGVHD, n = 15/JQ5). (B) Similar results are shown for each population with JQ1 treatment, consistent with reduced GC reaction (n = 14/BM-only, n = 11/cGVHD, n = 7/JQ1). (C) Representative gating for (left) TFH and TFR cells and (right) GCB cells. BM-only contours are in blue, cGVHD contours are in red. (D) Representative images of cryopreserved spleen sections stained to show GCs from mice 49 days posttransplant. Sections are stained with DAPI (blue), peanut agglutinin (PNA) rhodamine (red), and CD4 FITC (green). Images are at ×200 magnification. (E-F) (Left) Number of GCs observed in each spleen section normalized for the area of spleen in each section. A GC was counted if it was a roughly circular region of PNA+ cells near a region of CD4+ cells. Right: quantification of the average size of GCs observed in each section. The GC size was determined as the area of the PNA+ region. For both JQ5 (E; n = 8/BM-only, n = 6/cGVHD, n = 5/JQ5) and JQ1 (F; n = 5/group), there was a significant reduction in both GC count and average size. (G) Flow cytometric analysis of single-cell lung suspensions taken from transplanted mice treated with each drug (n = 5/group). Left: total plasma cells are CD138+ lymphocytes, (center) immature plasma cells are B220+, CD19+ plasma cells, and (right) mature plasma cells are B220−, CD19− plasma cells. Results show that both drugs significantly reduced the proportion of mature plasma cells in subject lungs. (H) Mean fluorescence intensity (MFI) quantification of flow cytometry analysis of H3K27me3 content of GC cell populations gated as in panel C (n per group as in panel A). (I) Representative histograms of GCB cells H3K27me3 content for BM-only, cGVHD, and JQ5-treated mouse-derived cells. For all panels, statistics shown are results of an unpaired t test with Bonferroni corrected P values, where appropriate. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal